Cloning and characterization of the biosynthetic gene cluster for kutznerides Danica Galonic´Fujimori†, Sinisˇa Hrvatin†, Christopher S. Neumann†, Matthias Strieker†‡, Mohamed A. Marahiel‡, and Christopher T. Walsh†§ †Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA 02115; and ‡Department of Chemistry/ Biochemistry, Philipps University, 35032 Marburg, Germany Contributed by Christopher T. Walsh, August 30, 2007 (sent for review August 2, 2007) Kutznerides, actinomycete-derived cyclic depsipetides, consist of halogenating species (6). Because chloride is transferred as a Clϩ six nonproteinogenic residues, including a highly oxygenated equivalent, only electron-rich carbon centers in substrates can be tricyclic hexahydropyrroloindole, a chlorinated piperazic acid, 2-(1- chlorinated. By contrast, chlorination of unactivated carbon methylcyclopropyl)-glycine, a ␤-branched-hydroxy acid, and 3- centers is carried out by the class of nonheme iron enzymes hydroxy glutamic acid, for which biosynthetic logic has not been represented by the syringomycin halogenase SyrB2 (7), the elucidated. Herein we describe the biosynthetic gene cluster for barbamide halogenases BarB1 and BarB2 (8), and the aliphatic the kutzneride family, identified by degenerate primer PCR for halogenase CytC3 (9). These enzymes chlorinate an unactivated halogenating enzymes postulated to be involved in biosyntheses methyl group of protein thiolation (T)-domain-tethered amino of these unusual monomers. The 56-kb gene cluster encodes a acids and require Fe(II), oxygen, chloride, and ␣-ketoglutarate series of six nonribosomal peptide synthetase (NRPS) modules for activity. Substrate activation is achieved by hydrogen atom distributed over three proteins and a variety of tailoring en- abstraction by an Fe(IV)-oxo species (10), resulting in a HO- zymes, including both mononuclear nonheme iron and two flavin- Fe(III)-Cl intermediate that transfers a chlorine atom to the dependent halogenases, and an array of oxygen transfer catalysts. substrate radical, resulting in the formation of product. Studies The sequence and organization of NRPS genes support incorpora- on the biosynthesis of coronamic acid (1-amino-1-carboxy-2- tion of the unusual monomer units into the densely functionalized ethyl cyclopropane) showed that the cyclopropane moiety is scaffold of kutznerides. Our work provides insight into the for- formed via a cryptic chlorination pathway (11). A T-domain- mation of this intriguing class of compounds and provides a tethered ␥-chloro-allo-Ile thioester is the substrate for the foundation for elucidating the timing and mechanisms of their cyclopropane-forming enzyme CmaC, which catalyzes the in- biosynthesis. tramolecular displacement of chloride by a thioester-stabilized aminoacyl ␣-carbanion (12). Combined, these studies demon- chlorination ͉ halogenases ͉ nonribosomal peptide biosynthesis strated that halogenating enzymes not only account for the production of chlorinated secondary metabolites in bacteria but utznerides are antifungal and antimicrobial cyclic hexadep- also are involved in the formation of chlorinated intermediates Ksipeptides isolated from the soil actinomycete Kutzneria sp. 744 during the biosynthesis of cyclopropane residues. (1). Structural elucidation of these metabolites revealed nine re- We postulate that the MecPGly residue in kutznerides could lated compounds composed of five unusual nonproteinogenic arise from a cryptic chlorination pathway involving such a amino acids and one hydroxy acid (Fig. 1) but differing in the extent nonheme iron halogenase, although through a different enzy- of substitution and stereochemistry of constituent residues (2). All matic logic to that of CmaC-catalyzed cyclopropane formation, kutznerides contain 2-(1-methylcyclopropyl)-D-glycine (MecPGly) given the attachment of the cyclopropane ring to C␤, rather than connected to the ␣-hydroxyl moiety of either (S)-2-hydroxy-3- the C␣ of the amino acid. Furthermore, the presence of two methylbutyric or (S)-2-hydroxy-3,3-dimethylbutyric acid. The hy- halogenated residues, 6,7-dichlorinated hexahydropyrroloindole droxy acid residue is followed by a piperazic acid moiety, found in in all isolated kutznerides and 4-chloropiperazate in kutznerides four distinct forms: as piperazic acid in kutznerides 1, 3, 5, and 7; 2 and 8, implicates flavin-dependent halogenases in kutzneride as dehydropiperazate in kutznerides 4 and 9; as ␥-chloro-piperazate biosynthesis. We hypothesized that the gene cluster responsible in kutznerides 2 and 8; and as ␥-hydroxy-dehydropiperazate in for the biosynthesis of these secondary metabolites could be kutzneride 6. Furthermore, kutznerides contain O-methyl-L-serine identified by degenerate primer-based PCR amplification of and either the threo or erythro isomer of 3-hydroxy-D-glutamate. highly conserved mononuclear nonheme iron (9, 13, 14) and Finally, an unusual tricyclic dihalogenated (2S,3aR,8aS)-6,7- flavin-dependent halogenase sequences (15–18) in a cosmid dichloro-3a-hydroxy-hexahydropyrrolo[2,3-b]indole-2-carboxylic library constructed from genomic DNA. Herein, we use this acid (PIC) is conserved in all structurally characterized kutznerides. strategy to find the biosynthetic gene cluster of kutznerides, The structural subunits of kutznerides suggest unusual enzymatic mechanisms involved in their biosynthesis. Recently, our laboratory has elucidated the enzymatic logic of Author contributions: D.G.F., S.H., C.S.N., M.S., and C.T.W. designed research; D.G.F., S.H., carbon–chlorine bond formation during the biosynthesis of C.S.N., and M.S. performed research; D.G.F., S.H., C.S.N., M.S., and C.T.W. analyzed data; several chlorinated secondary metabolites. Among these, dichlo- and D.G.F., S.H., C.S.N., M.S., M.A.M., and C.T.W. wrote the paper. rination of the pyrrole moiety in the biosynthesis of pyoluteorin The authors declare no conflict of interest. (3) and chlorination of tryptophan in rebeccamycin biosynthesis Abbreviations: MecPGly, 2-(1-methylcyclopropyl)-D-glycine; NRPS, nonribosomal peptide synthetase; T domain, thiolation domain; KR, ketoreductase domain; E domain, epimer- (4) are carried out by the flavin-dependent halogenases PltA and ization domain; PPi, pyrophosphate. RebH, respectively. The work of van Pee and coworkers (5) on Data deposition: The sequence reported in this paper has been deposited in the GenBank the pyrrolnitrin halogenase PrnA demonstrated the necessity of database (accession no. EU074211). FADH2, chloride, and oxygen for catalytic activity of flavopro- §To whom correspondence should be addressed. E-mail: christopher࿝[email protected]. tein halogenases. The addition of chloride to the terminal oxygen edu. of a FAD-C4a-OOH intermediate in these enzymes generates This article contains supporting information online at www.pnas.org/cgi/content/full/ reactive hypochlorous acid, which is proposed to react with the 0708242104/DC1. enzyme’s active-site lysine to form lysine chloramine as the active © 2007 by The National Academy of Sciences of the USA 16498–16503 ͉ PNAS ͉ October 16, 2007 ͉ vol. 104 ͉ no. 42 www.pnas.org͞cgi͞doi͞10.1073͞pnas.0708242104 Downloaded by guest on September 29, 2021 dioxygenases, a flavin-dependent oxygenase and a cytochrome P450. Bioinformatic analysis of the function of NRPS modules and modifying enzymes has allowed for prediction of enzymatic steps leading to the biosynthesis of the unusual building blocks and their order of utilization during chain growth. The identity of the cluster has been confirmed via the analysis of amino acid activation specificity of three of the seven constituent adenyla- tion domains. Results and Discussion Identification of Kutzneride Cluster from Kutzneria sp. 744. The producing organism that yields kutznerides 1–9 was cultivated as described (1) and its genomic DNA isolated. Amplicons ob- tained by PCR using genomic DNA as a template and degen- erate primers for both flavin and mononuclear nonheme iron halogenases [supporting information (SI) Table 2] were se- quenced, identifying two distinct flavin-dependent halogenases and one Fe(II)-dependent halogenase. Specific primers were thereby designed (SI Table 2) and used to screen a cosmid library of genomic DNA. The cosmid library was prepared by cloning 30- to 50-kb fragments followed by lambda-phage transduction and preparation of 50 liquid gel culture pools (19), each har- boring Ϸ60 colonies. Positive pools identified with specific primers were plated and positive colonies identified. No single colony harboring both nonheme iron and the two flavin halo- genase genes was identified. Further PCR screening with prim- ers specific for ends of DNA inserts identified two colonies with sequence overlap, one containing genes for both flavin haloge- nases and the other with the nonheme iron halogenase. The two inserts from these colonies were fully sequenced (Agencourt Fig. 1. Structures of kutznerides 1–9. Bioscience, Beverley, MA). Analysis of Kutzneride Gene Cluster. The biosynthetic gene cluster which reveals that these hexadepsipeptides are biosynthesized on of kutznerides spans Ϸ56 kb of genomic DNA and consists of 29 a modular nonribosomal peptide synthetase (NRPS) assembly ORFs, 17 of which can be assigned roles in kutzneride biosyn- BIOCHEMISTRY line. The most striking feature of the gene cluster is the large thesis (Fig. 2A and Table 1). In analogy to other bacterial number of genes encoding for tailoring enzymes that carry out secondary metabolites